Methods and compositions including a 13 kD B. burgdorferi protein

ABSTRACT

All  Borrelia burgdorferi sensu lato  isolates characterized to date have one or a combination of several major outer surface proteins (Osp). Mutants of  B. burgdorferi  lacking Osp proteins were selected with polyclonal or monoclonal antibodies at a frequency of 10 −6  to 10 −5 . One mutant that lacked OspA, B, C and D was further characterized in the present study. It was distinguished from the OspA + B +  cells by its (i) auto-aggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum- and complement-sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in the skin the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two different classes, A and B, of monoclonal antibodies (mAb) with growth inhibiting properties for mutant cells were produced. Class A mAbs bound to 13 kDa surface proteins of  B. burgdorferi sensu stricto  and of  B. afzelii . The minimum inhibitory concentration of the Fab fragment of one mAb of this class was 0.2 μg/ml. Class B mAbs did not bind by Western Blot to a  B. burgdorferi  cells but reacted with cells in an unfixed cell immunofluorescence assay and growth inhibition assay. These studies revealed hitherto unknown functional aspects of Osp proteins, notably serum-resistance, and indicated that in the absence of Osp proteins other antigens are expressed or become accessible at the cell&#39;s surface.

This application is a divisional of U.S. application Ser. No.08/264,036, filed Jun. 22, 1994, now U.S. Pat. No. 6,300,101 issued Oct.9, 2001, which is a continuation-in-part of application Ser. No.08/079,601, filed Jun. 22, 1993, now U.S. Pat. No. 5,523,089, which is acontinuation of U.S. application Ser. No. 07/924,798, filed Aug. 6,1992, now abandoned, which is a continuation of U.S. application Ser.No. 07/422,881, filed Oct. 18, 1989, now abandoned, claiming priorityfrom Danish application 5902/88, filed Oct. 24, 1988.

U.S. application Ser. No. 08/264,036 is also a continuation-in-part ofU.S. Ser. No. 08/124,771, filed Sep. 21, 1993, now abandoned; which wasa continuation-in-part of U.S. Ser. No. 07/781,355, filed Oct. 22, 1991,now issued as U.S. Pat. No. 5,246,844.

The United States Government has certain rights in the present inventionpursuant to grants AI-29731 and AI-26804 from the National Institutes ofAllergy and Infectious Diseases.

BACKGROUND OF THE INVENTION

Lyme disease is a complex, multisystemic illness caused by at leastthree genomic species of the spirochete Borrelia burgdorferi sensu lato(reviewed in Barbour and Fish, 1993). Virtually all North Americanisolates have been classified as B. burgdorferi sensu stricto (Barantonet al., 1992; Boerlin et al., 1992; Welsh et al., 1992). Europeanisolates also include two other genomic species, B. garinii and B.afzelii (Baranton et al., 1992; Canica et al., 1993). The clinicalfeatures and epidemiology of Lyme disease have been well characterized(reviewed review in Barbour and Fish, 1993). Comparatively less,however, is known about the pathogenic features of Lyme diseaseborrelias and immunopathological responses to them in the host.

Ignorance of precise mechanisms of Lyme disease pathogenesis is partlyattributable to the paucity of basic information about all spirochetes.The spirochete cell is unique in several aspects (Holt, 1978). One ofthe features of borrelia is the abundance of one or several lipoproteinsin the outer cell membrane (Bergstrom et al., 1989; Brandt et al., 1990;Brusca et al., 1991; Howe et al., 1985; Norris et al., 1992). Much hasbeen learned about immunogenicity, as well as biochemical and geneticaspects, of these lipoproteins in Lyme disease and relapsing feverborrelias (Barbour, 1993; Bergstrom et al., 1989; Brandt et al., 1990;Johnson et al., 1992; Kitten and Barbour, 1990; Meier et al., 1985;Wilske et al., 1993).

The lipoproteins OspA and OspB are major contributors to antigenicdistinctness of Lyme disease borrelias (Barbour and Fish, 1993). BothOspA and OspB are co-transcribed from a single operon located on linearplasmid of 49 kb in B. burgdorferi sensu stricto (Bergstrom et al.,1989). Many of European and some North American B. burgdorferi sensulato strains express a third immunodominant major protein, OspC (Wilskeet al., 1993). Another protein of this group, OspD, has been alsoreported (Norris et al., 1992). Proteins called “OspE” and “OspF” havebeen reported, but their surface exposure and location in the outermembrane have not been established (Lam et al., 1994).

OspA and OspB may contribute to the spirochete's ability to adhere to orinvade host cells (Benach et al., 1988; Comstock et al., 1992; Thomasand Comstock, 1989). It has been suggested that OspA may affect thechemotactic response of human neutrophils in vitro (Benach et al.,1988). Mitogenic and cytokine-stimulatory properties of OspA and OspBhave been also shown (Ma and Weis, 1993). It was found that reduced sizeand amounts of OspB was associated with lowered infectivity (Sadziene etal., 1993A). The findings of Cadavid et al. indicated that differencesin invasive properties and tissues tropism between serotypes of relatedspirochete Borrelia turicatae, a relapsing fever agent, may bedetermined by the expression of a single surface protein that isanalogous to Osp proteins of B. burgdorferi (Cadavid et al., 1994).

These studies of function of Osp proteins, however, are still limited innumber. More information is needed regarding the function of theseproteins, in particular their roles in infectivity and theircontributions to the microorganism's ability to survive in the host. Oneapproach to obtain these insights is selection and characterization ofmutants with altered surface lipoproteins. There were several compellingreasons for studying B. burgdorferi cells that lacked all known Ospproteins (Sadziene et al., 1992, Sadziene et al., 1993B). First themorphology and function of the Osp-less mutant were characterized todetermine whether borrelias lacking OspA, B, C, and D would be alteredin such functional properties, as (i) generation time, (ii) ability toform colonies on solid medium, (iii) adherence to cells, (iv) serum andcomplement sensitivity, (v) potential to evoke immune response afterintradermal live cell inoculation, and (vi) ability to survive in theskin. Among pathogenic borrelias the role of surface lipoproteins inthese respects have not yet been reported.

Another intriguing aspect was the immunological characterization of theOsp-less mutant. There have been several studies describing lowmolecular weight lipoproteins that have not been identified as Osps.Katona et al. showed the presence of a major low-molecular-weightlipoprotein specific for B. burgdorferi and raised the possibility thatit was a borrelial equivalent of Braun's lipoprotein (Katona et al.,1992). Another study reported an immunogenic 14 kDa surface protein ofB. burgdorferi recognized by sera from Lyme disease patients (Sambri etal., 1991). These findings encouraged us to determine whether otherproteins are present on the surface in the absence of Osp proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Phase contrast photomicrograph of aggregation of B. burgdorferi313 by monoclonal antibodies. 4+ aggregation (see Methods) by class Aantibody.

FIG. 1B. Phase contrast photomicrograph of aggregation of B. burgdorferi313 by monoclonal antibodies. 3+ aggregation by class B antibody. Bar,1.0 μm.

FIG. 2. Coomassie blue-stained polyacrylamide gel (CB) and Western blotanalysis (WB) of whole-cell lysates of B. burgdorferi isolates B31,B311, B312, B313, and B314 with either monoclonal antibody 15G6 and 7D4.The acrylamide concentration was 17%. The molecular size standards(×1000) were ovalbumin, carbonic anhydrase, β-lactoglobulin) lysozyme,and bovine trypsin inhibitor.

FIG. 3A. Western blot analysis with antibody 15G6. B. burgdorferi B311and B313, B. afzelii ACAI, B. garinii IP90 and B. hermsii Bh33 wereprobed with the antibody 15G6 mAb.

FIG. 3B. B313 cells treated (+) or untreated (−) with proteinase K (PK).The molecular size standards (×1000) were carbonic anhydrase,β-lactoglobulin, lysozyme, and bovine trypsin inhibitor.

FIG. 4A. Binding of fluorescein-conjugated monoclonal antibody 15G6 toB. burgdorferi B313. Left-upper and right-upper panels, directimmunofluorescence of unfixed cells in suspension for 3 min (left) and15 min (right). Lower panel, phase contrast photomicrograph ofaggregates and membrane blebs (arrow head). Bar. 10 μm.

FIG. 4B. Binding of fluorescein-conjugated monoclonal antibody 15G6 toB. burgdorferi B313. Left-upper and right-upper panels, directimmunofluorescence of unfixed cells in suspension for 3 min (left) and15 min (right). Lower panel, phase contrast photomicrograph ofaggregates and membrane blebs (arrow head). Bar, 10 μm.

FIG. 4C. Binding of fluorescein-conjugated monoclonal antibody 15G6 toB. burgdorferi B313. Left-upper and right-upper panels, directimmunofluorescence of unfixed cells in suspension for 3 min (left) and15 min (right). Lower panel, phase contrast photomicrograph ofaggregates and membrane blebs (arrow head). Bar, 10 μm.

FIG. 4D. Binding of fluorescein-conjugated monoclonal antibody 15G6 toB. burgdorferi B313. Left-upper and right-upper panels, directimmunofluorescence of unfixed cells in suspension for 3 min (left) and15 min. (right). Lower panel, phase contrast photomicrograph ofaggregates and membrane blebs (arrow head). Bar, 10 μm.

FIG. 5A. Phase contrast photomicrographs (left) and directimmunofluorescence (right) of unfixed B. burgdorferi in suspension. A(upper) panels, B313 cells with fluorescein-conjugated antibody 15G6. B(lower) panels, B311 cells with unconjugated antibody H6831 andconjugated antibody 15G6. 15G6 alone did not bind to B311 cells (notshown). Bar, 2.0 μm. FIG. 5 has four panels, two in A and two in B; andno further panels or drawing elements.

FIG. 5B. Phase contrast photomicrographs (left) and directimmunofluorescence (right) of unfixed B. burgdorferi in suspension. A(upper) panels,. B313 cells with fluorescein-conjugated antibody 15G6. B(lower) panels, B311 cells with unconjugated antibody H6831 andconjugated antibody 15G6. 15G6 alone did not bind to B311 cells (notshown). Bar, 2.0 μm. FIG. 5 has four panels, two in A and two in B; andno further panels or drawing elements.

FIG. 5C. Phase contrast photomicrographs (left) and directimmunofluorescence (right) of unfixed B. burgdorferi in suspension. A(upper) panels, B313 cells with fluorescein-conjugated antibody 15G6. B(lower) panels, B311 cells with unconjugated antibody H6831 andconjugated antibody 15G6. 15G6 alone did not bind to B311 cells (notshown). Bar, 2.0 μm. FIG. 5 has four panels, two in A and two in B; andno further panels or drawing elements.

FIG. 5D. Phase contrast photomicrographs (left) and directimmunofluorescence (right) of unfixed B. burgdorferi in suspension. A(upper) panels, B313 cells with fluorescein-conjugated antibody 15G6. B(lower) panels, B311 cells with unconjugated antibody H6831 andconjugated antibody 15G6. 15G6 alone did not bind to B311 cells (notshown). Bar, 2.0 μm. FIG. 5 has four panels, two in A and two in B; andno further panels or drawing elements.

FIG. 5E. Phase contrast photomicrographs (left) and directimmunofluorescence (right) of unfixed B. burgdorferi in suspension. A(upper) panels, B313 cells with fluorescein-conjugated antibody 15G6. B(lower) panels, B311 cells with unconjugated antibody H6831 andconjugated antibody 15G6. 15G6 alone did not bind to B311 cells (notshown). Bar, 2.0 μm. FIG. 5 has four panels, two in A and two in B; andno further panels or drawing elements.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS MATERIALS AND METHODS

Strains and Culture Conditions

B. burgdorferi sensu stricto mutants were of the B31 (ATCC 35210)lineage (Table 1). The Osp phenotypes and plasmid contents ofnoninfectious derivatives B311, B312, B313 and B314 were describedpreviously under these or other designations (Barbour, 1984; Barbour andGaron, 1987; Hinnebusch and Barbour, 1992; Sadziene et al., 1993B).Populations that were passed in medium not more than 10 times wereconsidered low passage isolates. The low passage, infectious progenitorfor this lineage retained the original strain designation, B31(Burgdorferi et al., 1982). With the exception of B31, all cells of thislineage were grown from single cell clones. In some experiments otherstrains were used: HB19 (Barbour et al., 1983; Steere et al., 1983) andSh.2 (Schwan et al., 1988), both of which are B. burgdorferi sensustricto, B. afzelii strain ACAI (Boerlin et al., 1992) and B. gariniistrain Ip90 (Baranton et al., 1992; Boerlin et al., 1992) (Table 1). B.hermsii HS1 serotype 33 (ATCC 35209; Barbour et al., 1982) wasabbreviated to Bh33. Borrelias were grown in BSK II medium and harvestedby methods described previously (Barbour, 1984; Barbour et al., 1983).When culturing tissues from animals, rifampicin (50 μg/ml), phosphomycin(100 μg/ml) and, for skin samples, additionally amphotericin (25 μg/ml)were added to the medium. Cells were counted in a Petroff-Hauser chamberby phase-contrast microscopy. In some studies borrelias were also grownon solid BSK II medium as described (Hinnebusch and Barbour, 1992;Sadziene et al., 1992). To estimate growth rate, borrelias at an initialconcentration of 2×10⁶ cells/ml, were grown in tightly capped, 13×100-mmpolystyrene culture tubes (Falcon Labware, Lincoln Park, N.J.)containing 6 ml of medium. Growth at 34° C. in 1% CO₂ atmosphere wasmonitored visually and by cell counts every 12 h for 3 d. The amount oftotal cellular protein in the final cell pellet was determined withBradford reagent (Bio-Rad Laboratories, Richmond, Calif., (Barbour etal., 1983). The microscopic aggregation of borrelias alone or in thepresence of antibodies was graded according to the following scale: 0,single cells with less than 10% of the cells in clumps of 2-10 cells;1+, 10-50% of cells in clumps of 2-10, 2+, 10-50% of cells in clumps of11-100; 3+, >50% of cells in clumps of 11-100; and 4+, >50% of cells inclumps of >100.

TABLE 1 ISOLATES OF B. BURGDORFERI SENSU LATO USED IN THE STUDY ANDTHEIR OSP PROFILE Genomic Osp profile^(a) species Isolate OspA OspB OspCOspD Reference B. burgdorferi B31 + + − + Burgdorfer et al. 1982;Sadziene et al. 1993B B311 + + − − Barbour, 1984; Barbour and Garon,1987; Sadziene et al, 1993B. B312 + + + − Hinnebusch and Barbour, 1992;Sadziene et al, 1993B. B313 − − − − Sadziene et al, 1992, 1993b. B314 −− + − Sadziene et al, 1993B. HB19 + + + + Barbour et al, 1983.; Steereet al., 1983 Sh.2 + + + − Schwan et al., 1988 B. afzelii ACAI + + + −Boerlin et al., 1992 B. garinii IP90 + + + − Baranton et al., 1992;Boerlin et al., 1992 ^(a)Osp profile was determined by Western blotanalysis.

Antisera and Monoclonal Antibodies (mAbs)

The origins of the OspA-specific mAb H5332 (Barbour et al., 1983),OspB-specific mAb H6831 (Barbour et al., 1984) and Vmp33-specific mAbH4825 (Barbour et al., 1984) have been given. Monoclonal antibody H9724binds to native and denatured flagellins of different Borrelia species(Barbour et al., 1986). These antibodies are IgG subclass 2a (IgG2a).

Additional polyclonal and monoclonal antibodies were produced for thisstudy. Female, 6-8 week old BALB/c mice (Jackson Laboratory, Bar Harbor,Me.) were used. Freshly-harvested borrelias were washed with andresuspended in PBS, pH 7.0. The total cellular protein in the suspensionwas estimated with Bradford reagent and adjusted with PBS for a totalprotein concentration of 200 μg/ml. 0.5 ml of antigen suspension wasemulsified in 0.5 ml of complete Freund's adjuvant (CFA; Sigma ChemicalCo., St. Louis, Mo.), and 200 μl of emulsion was administered as sixsubcutaneous injections at day 0. Control mice received a 200 μlemulsion of equal parts of CFA and PBS alone. The total dose per mousewas 20 μg protein. After 4 weeks mice were boosted with the same dose.Mice were bled by eye sinus puncture 10 days after the boost. Aftercollection, sera were evaluated by ELISA and GIA. On day 52, the micereceived intravenously 2×10⁸ viable borrelias in 100 μl of PBS. Fusionof mouse splenocytes with NS1 myeloma cells were performed on day 56 bya modification of the previous method (Oi and Herzenberg, 1980).Undiluted hybridoma supernatant fluids without antibiotics were screenedby wet ELISA, unfixed cell IFA and Western Blot techniques. Those fluidsthat were positive by either one of these methods were then evaluated byGIA. For GIA hybridoma supernatant fluids were dialyzed against PBS, pH7.0 and concentrated with Centriprep®-10 (Amicon, Beverly, Mass.)cartridges. The isotypes of antibodies were determined using acommercial kit (Immunotype™; Sigma Chemical Co., St. Louis, Mo.).Ascitic fluids from hybridomas were produced as described (Sadizene etal., 1994).

Purified mAbs and univalent Fab fragments were prepared from hybridomasupernatants essentially as described (Sadziene et al., 1993C). Briefly,hybridoma supernatants were concentrated using an Amicon 8200 membraneconcentrator with a Diaflo® YM30 ultrafiltration membrane (Amicon) under50 psi N₂. Purified mAbs were obtained by Protein A-sepharose columnchromatography. Univalent Fab fragments were prepared using theImmunopure® Fab Preparation kit (Pierce Chemical Co.) by cleaving thepurified antibodies with papain, retaining intact immunoglobulin and Fcfragments on a protein A-sepharose column, and dialyzing the void volumeof the column against PBS, pH 7.0. Purified mabs and Fab fragments wereconcentrated with Centriprep®-10 (Amicon). Protein concentrations weredetermined by UV spectrophotometry at 280 nm. Purified whole IgG and Fabfragments were analyzed by SDS-PAGE. Reactivities of purified mAbs andFab fragments were confirmed by direct and indirect immunofluorescenceassay, Western blot and GIA.

Elisa

The method for ELISA was essentially as described previously (Sadzieneet al., 1991). For this “dry” ELISA borrelias at a total proteinconcentration of 1.4 μg/ml in phosphate-buffered saline (PBS), pH 7.0were dried onto polystyrene 96-well microtiter plates at 37° C. for 18h. For a “wet” ELISA borrelias at a total protein concentration of 3μg/ml in 15 mM Na₂CO₃—35 mM NaHCO₃ buffer, pH 9.6 were coated ontoplates at 4° C. for 24 h. After blocking for 1 h at 37° C. with 1%(wt/vol) dried nonfat milk in PBS (milk/PBS) and washing with PBS alone,twofold dilutions of antibody in milk/PBS were added. The plates wereincubated for 2 h at 37° C. and washed with PBS. Bound antibody wasmeasured using alkaline phosphatase-conjugated goat anti-mouse IgG(Zymed). The substrate was p-nitrophenyl phosphate (Sigma). Absorbancevalues were recorded at 490 nm on a model 580 ELISA reader (DynatechLaboratories, Chantily, Va.); wells with values ≧0.2 were consideredpositive.

Immunofluorescence Assays

Indirect immunofluorescence assay (IFA) of fixed, dried cells wasperformed as described (Barbour et al., 1982; Barbour et al., 1983).Harvested, fresh borrelias were washed with RPMI 1640 medium, mixed witha suspension of washed rat erythrocytes in 50% RPMI 1640—50% fetal calfserum, and a thin smear of the suspension was coated on the slides.Slides were fixed in methanol, air dried, and kept in a dessicator at−20° C. until use.

Binding of mAb to unfixed live spirochetes was assessed by amodification of the described procedure (Barbour et al., 1983). 10⁷borrelias were washed with 2% (wt/vol) BSA in PBS/Mg (PBS/Mg/BSA) andthen resuspended in 0.5 ml of undiluted hybridoma culture supernatant or0.5 ml of PBS/Mg/BSA containing the mAb of interest. The cell mixturewas incubated at room temperature with gentle rotation for 60 min. Thecells were centrifuged, washed twice with PBS/Mg/BSA, resuspended in 30μl volume of PBS/Mg/BSA with 20 μg/ml of anti-mouse Ig-fluoresceinF(ab′)₂ fragment (Boehringer-Mannheim, Indianapolis, Ind.) and incubatedfor 30 min under the same conditions. Before microscopic evaluation thevolume of the cell suspension was adjusted to 300 μl with PBS/Mg/BSA.

For direct IFA purified mAbs and their Fab fragments were conjugatedwith fluorescein isothiocyanate (QuickTag FITC Conjugation Kit;Boehringer-Mannheim). Fractions containing the antibody-fluoresceinconjugate were mixed together, dialyzed in the dark against PBS for 24h, and concentrated with a Centriprep®-10 (Amicon, Beverly, Mass.). 10⁷borrelias in log-phase growth were resuspended in RPMI 1640 medium with10-100 μg/ml of antibody-fluorescein conjugate and examined forfluorescence at 3, 15, 30, 60, and 360 min.

Growth Inhibition Assays

The growth inhibition assay (GIA) was described previously (Sadziene etal., 1993C). Briefly, to a 100 μl volume of BSK II containing 2×10⁶borrelias was added an equal volume of heat-activated (56° C. for 30min) mAb or polyclonal antiserum, serially diluted two-fold in BSK II.To evaluate the susceptibility of borrelias to fresh, nonimmune serum,the same growth inhibition technique was applied using pooled unheatedserum from C3H/HeN mice (Taconic, Germantown, N.Y.). Blood was drawn onice, separated from red blood cell clot, and immediately frozen at −135°C. Heat-inactivated serum from the same mice served as a control. Todetermine the susceptibility of borrelias to complement, unheated orheated (56° C. for 30 min) guinea pig complement (Diamedix, Miami, Fla.)was added to each well at an activity ranging from 6 to 1 hemolytic unit(HU; CH₅₀) per well. In some experiments, 2 HU of unheated guinea pigcomplement were added to each well for a final concentration of 10 HU/mlof medium after addition of antibody.

The incubations were performed in flat-bottomed, 96-well, polystyrenemicrotiter plates, covered by adhesive, clear plastic seals (SensititreMicrobiologic Systems, Westlake, Ohio) and were carried out for 72 h at34° C. in a 1% CO₂ atmosphere. Growth in the wells was monitoredvisually for changes in the color of the phenol red indicator and byphase contrast microscopy of wet-mounts of culture samples. A pink colorof the indicator after incubation represented at least 20-fold fewercells in these wells than in wells that were yellow. The minimalinhibitory concentration (MIC) was the lowest concentration of mAb thatproduced pink instead of yellow wells (Sadziene et al., 1993C). Allgrowth inhibition studies were performed at least twice.

Electrophoresis and Western Blot Analysis

Whole-cell lysates were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PACE) with 15% or 17%acrylamide as described previously (Barbour, 1984; Barbour et al.,1982). In some experiments, cleavage of surface-exposed proteins ofintact borrelias with proteinase K (Boehringer-Mannheim) was carried out(Sadziene et al., 1992). For this study 490 μl of a suspensioncontaining 5×10⁸ cells in PBS/Mg was mixed with 10 μl of proteinase Ksolution (20 mg/ml of water) and incubated for 40 min at 22° C. Thereaction was stopped by the addition of phenylmethylsulfonyl fluoride.

For Western blot analysis, proteins were transferred to nitrocellulosemembranes, which were then blocked with 3% (wt/vol) dried nonfat milk in10 mM Tris-HCl (pH 7.4)—150 mM NaCl (milk/TS) for 2 h as describedbefore (Oi and Herzenberg, 1980). After a wash in milk/TS, membraneswere incubated with mAb ascitic fluid diluted 1:50 or 1:100 in milk/TSor hybridoma supernatant fluid diluted 1:5 or 1:10 in milk/TS. Alkalinephosphatase-conjugated recombinant protein A/G (Immunopure; PierceChemical Co., Rockford, Ill.) served as the second ligand. The blotswere developed with nitro-blue tetrazolium chloride and5-bromo-4-chloro-3-indolylphosphatase p-toluidine salt (Pierce,Rockford, Ill.).

Adherence Assay

An assay for adherence of intrinsically-labeled borrelias to humanumbilical vein endothelium (HUVE) cells was carried out essentially asdescribed (Thomas and Comstock, 1989). Briefly, borrelias wereintrinsically radiolabeled with [³⁵ S]-methionine, washed with PBS andresuspended to a density of 1.7×10⁸ cells per ml in Medium 199 with 20%fetal calf serum. 300 μl aliquots of radiolabeled spirochetes were addedto confluent HUVE cell monolayers grown in 24-well plates. After a 4 hrincubation at 4° C., monolayers with associated organisms were washed,solubilized, mixed with scintillation cocktail (Universol ES; ICNPharmaceuticals, Irvine, Calif.), and counted by scintillation. Theassay was done with triplicate samples and performed twice. Differencesbetween borrelia populations in adhesion were analyzed by Student's ttest.

Experiments in Mice

Six-to-eight week old, female C3H/HeN mice (Taconic, Germantown, N.Y.)were used. Borrelias were counted and diluted in BSK II to give thedesired inoculum. For live cell immunization, 10 μl of cells in BSK IImedium was transferred to 900 μl of sterile PBS solution immediatelyprior to immunization. 100 μl of this suspension then was inoculatedintradermally in the abdomen at day 0. As a control, 100 μl of 0.1× BSKII in PBS was used. On day 24 mice were bled from the tail vein, andtheir sera were examined by ELISA and GIA. Mice were challenged on day28 at the base of the tail with 10⁴ of B. burgdorferi strain Sh.2(Sadziene et al., 1993A). Mice were euthanized 14 d following infection.Plasma (0.5 ml) obtained from citrated blood, the whole bladder,macerated heart, and cross-cuttings of both tibiotarsal joints wereadded to BSK II medium and cultured at 34° C. Cultures were examined forthe presence of motile spirochetes by phase-contrast microscopy at days7 and 14 of cultivation; they were scored as negative when no motilespirochetes were seen in forty 400× fields. For evaluation of borreliasurvival in skin, borrelias were diluted in 1× BSK II The abdominal skinwas shaved, and 10⁷ borrelia cells were injected intradermally at 3 or 4separate locations. Mice were sacrificed at 0.25, 0.5, 2, 6, 9, 12, 18and 24 h after injection and samples of skin from the injection siteswere immediately cultured in BSK II medium at 34° C.

RESULTS

Isolate B313 of the B31 lineage of B. burgdorferi lacked OspA, B. C, D(Table 1; Sadziene et al., 1992; Sadziene et al., 1993B). This mutantwas selected from a clonal population of B31 under the selectivepressure of an anti-OspA mAb. Isolate B311's Osp profile wasOspA⁺B⁺C⁻D⁻. Mutants that lack both OspA and OspB were selected withpolyclonal or monoclonal antibodies directed against B. burgdorferi at afrequency of 10⁶-10⁻⁵ (Sadziene et al., 1992). The genetic basis for theOsp-less phenotype was loss of a 38 kb and 49 kb linear plasmids andretention of a 16 kb plasmid (Norris et al., 1992; Sadziene et al.,1992; Sadziene et al., 1993B).

Growth Rate

Osp-less mutant B313 was easily distinguishable from B311, as well asfrom other high-passage, Osp-bearing isolates of the B31 lineage, inbroth culture by its tendency to form microscopic aggregates. B313cultures had aggregation scores of 1+ or 2+, whereas B311 had a score of0. Another observed difference was the decreased ability of B313 cellsto turn the phenol red indicator yellow in the BSK II, even when theculture reached stationary phase. One possible explanation for this isthat metabolic activity of the Osp-less mutant was lower than that ofwild-type borrelias. Alternatively, the OspA⁻ OspB⁻ mutant may have aslower rate of growth than its parent B311 and, consequently, does notreach the same cell densities as wild-type borrelias at a particulartime point. To examine these possibilities the growth rates of B311 andB313 were determined and the amount of borrelia protein in the finalcell pellet was measured.

B311 and B313 cells were grown until stationary phase, that is, when nofurther growth occurred, was reached. Cell counts were determined every12 h in triplicate, and the log₁₀ of mean cell counts were plottedagainst time. At stationary phase B311 cultures had a cell density of1.5-2.0×10⁸ cells/ml and B313 cultures had a cell density of 4-5×10⁷,fourfold lower. Protein concentrations in the final B311 and B313 cellpellets were 0.65 mg and 0.16 mg, respectively, a finding consistentwith the cell counts. The mean generation time (±standard error of themean) of B311 cells was 6.6±0.1 h; the values for B313 cells were9.5±0.2 h, 50% slower. These findings indicated that the Osp-less cellsboth grew more slowly and achieved a lower final cell mass than didtheir Osp-bearing counterparts.

Plating Efficiency

Another biological characteristic of the Osp-less mutant was alsoevaluated, namely, its ability to grow as a colony on solid medium.Current procedures for cultivation of different low and high passage B.burgdorferi on solid medium yield efficiencies of plating between 50 and100% (Hinnebusch and Barbour, 1992; Sadziene et al., 1993A; Sadziene etal., 1992). In previous studies it was found that otherantibody-resistant variant populations of the B31 lineage could beplated with the same high efficiency (Sadziene et al., 1992). Anexception was the very low plating efficiency of mutant B314 (Table 1),which lacks all linear plasmids and has an OspA⁻B⁻C⁺D⁻ phenotype(Sadziene et al., 1993B). These data suggested that mutants with Osp⁻phenotype might also have a lesser ability to form colonies.

This study was performed twice, each time plating in triplicate 10¹-10⁶borrelias per plate. B311 cells grew as colonies with the expectedplating efficiency of 50%. The efficiency of B313 plating was 0.01%,more than a thousand-fold lower than for B311 cells under the sameconditions. Of three arbitrarily-chosen colonies of B311 that grew inbroth medium and were then subjected to SDS-PAGE, all retained theOsp-less phenotype.

Adherence to Endothelial Cells

Adherence of radiolabeled B. burgdorferi B311 and B313 cells to HUVEcell monolayers was measured after 4 h at 4° C. At this temperatureborrelias do not detectably enter endothelial cells and adherence ofcells becomes maximal by 4 h (Comstock and Thomas, 1989). The assay wasrepeated twice. Results of two studies are shown in Table 2. The abilityof Osp-less cells to adhere HUVE monolayer both times was only half thatof wild-type borrelias, a difference that was significant (P<0.001).

Serum and Complement Sensitivity

Wild-type B. burgdorferi is resistant to the nonspecific bactericidalactivity of nonimmune serum, in spite of classical and alternativecomplement pathway activation (Kochi and Johnson, 1987). It wasdetermined whether or not the borrelias' ability to resist thenonspecific bactericidal effects of complement might be attributable toOsp proteins. Accordingly, B311 cells and the Osp-less mutant were firstexposed to two-fold serially diluted fresh, naive mouse serum in a GIA.Heat-inactivated serum was applied in the same assay in parallel. Asexpected, B311 cells were resistant to the nonimmune serum; no growthinhibitory effect on the cells was observed at the lowest serum dilutionof 1:8. In contrast, the minimum inhibitory titer of nonimmune serumagainst Osp-less borrelias was 1:64. In wells with inhibited growth theB313 cells were nonmotile and had large

TABLE 2 ADHERENCE TO HUVE CELLS BY B311 AND B313 Adherence^(b) Cell Meancpm adhered Mean % of Experiment^(a) population ± SEM^(c) inoculumadhered^(d) I B311 14719 ± 134 5.0 B313 7360 ± 36 2.5 II B311 13447 ±92  5.7 B313 6801 ± 83 2.9 ^(a)The specific activities of inocula foradherence assays in experiments I and II were 2.9 × 10⁵ and 2.3 × 10⁵cpm, respectively. ^(b)Measured following incubation for 4 h at 4° C.^(c)Radioactivity bound to host cells following incubation and washing,expressed as the mean of three samples. ^(d)Differences between borreliapopulations in adhesion were analyzed by a Student's t test (P < 0.001).

membrane blebs. When heat-inactivated serum was applied to either B311or B313 cells, growth inhibition or these morphologic effects were notobserved at a serum dilution 1:8. These findings suggested thatcomplement affected the Osp-less cells.

To further evaluate the serum-susceptibility of the Osp-less mutant, theeffect of different activities of guinea pig complement on B311 and B313cells was compared. The dose of applied complement varied between 1-6 HUper well, and, as a control, the same doses of heat-inactivatedcomplement were used. The study was performed twice. Whereasheat-inactivated guinea pig complement had no growth inhibitory effecton either isolate at the doses of 6 HU or less per well, there weresubstantial differences in the effect of unheated complement on B313 andB311. As little as 1 HU of complement inhibited growth of B313; thisrepresented an MIC of ≦5 HU/ml. The corresponding MIC of unheatedcomplement for B3311 cells was ≧25 HU/ml.

The frequency of B313 cells surviving in the presence of complement wasalso estimated. Because of B313's poor growth on solid medium, the studywas performed in 96-well microtiter plates (Sadziene et al., 1992).5×10⁶ of B311 or B313 cells were exposed to 3 HU/tube of guinea pigcomplement for 6 h. After this time cell suspensions were diluted to theconcentration of complement less than 1 HU/tube and aliquoted in 200 μlvolumes to individual microtiter plate wells at inocula ranging between10⁰-10⁵ cells per well. Cells that were exposed to heat-inactivatedcomplement or no complement at all served as controls. The frequency ofcomplement-resistant mutants of B313 was calculated using tables of thePoisson distribution to be 3-6×10⁻⁵.

Of 11 complement-resistant B313 clones that were transferred to mediumwithout complement, only 6 proliferated. When these 6 cultures wereagain exposed to 3 HU of complement, all were as susceptible as theparent population. This suggested that if some changes had occurred inthe cell, they most likely represented a phenotypic change. When the 6cultures derived from the resistant populations were examined by PAGE,there was no discernable difference between them and the control B313protein profiles.

Survival of Borrelias in Skin

In the previous study it was shown that outer surface lipoproteins mighthave a role in protecting borrelias from one nonspecific host defense,namely, complement. Borrelias invade the host through the skin, beingable to survive in it from a few days to years (Steere, 1989).Accordingly, it was evaluated whether Osp proteins might also protectborrelias from nonspecific resistance factors in the skin of the mouse.,(e.g., different chemical substances from tissues with antibacterialactivity, early inflammation factors, and phagocytic cells) (Boyd andHoerl, 1986).

In a first step assessing these factors, it was determined how long B311and B313 cells would survive in the skin after intradermal inoculation.The study was repeated twice. In total 8 to 12 separate skin locationswere evaluated for spirochetal growth at the each time point. Mice weresacrificed at 0.25, 0.5, 2, 6, 9, 12, 18 and 24 h following inoculation,and full-depth skin biopsies were cultured. All cultures from up to 9 hwere positive with both B311 and B313. In 12 h, 4 out of 8 and 5 out of8 skin cultures were positive with B311 and B313 cells, respectively.None of the cultures from 18 and 24 h after inoculation was positive.These findings indicated that OspA and/or OSpB might not benefit theborrelias' survival in the skin. To confirm that cells that survived inthe skin retained the same phenotype, 6 randomly chosen cultures each ofB311 and B313 were subjected to SDS-PAGE; all of the examined cellsretained an unchanged protein profile.

Immunization by Intradermal Inoculation with Live Cells

The next study addressed whether live cells lacking known Osplipoproteins were able to induce immune response in the skin and, if so,how that response differed from the one induced by osp-bearing cells. Arationale for this study was the fact that viable (but noninfectious) B.burgdorferi of strain HB19 (at single intradermal dose of 10⁶ live cellsper mouse) were sufficient protect mice against challenge with 10⁴ Sh.2cells 4 weeks later (Sadziene and Barbour, 1994). This immunization dosewas used with B311 and B313 cells in the present study. The immuneresponses of immunized and control mice were evaluated by ELISA and GIAwith B311 and against the challenge strain, Sh.2.

As shown in Table 3 only immunization with cells expressing OspA andOspB, that is, B311, was effective in protecting all 5 mice fromexperimental infection with 10⁴ cells of the challenge strain. Osp-lessB313 failed to elicit a protective immune response at a immunizationdose of 10⁶ cells. All 5 mice that were immunized with live Osp-lessmutant cells, as well as control mice injected with 0.1× BSK alone,became infected. Immune responses among the groups as evaluated by ELISAand GIA also differed substantially.

Whereas B311 cells evoked an immune response as assessed by ELISA andespecially by GIA, the response to B313 cells in the same assays wassimilar to that of the control group. Western blot analysis with serafrom mice immunized with B313 showed no response to proteins of B.burgdorferi, except for faint bands against flagellin (Sadziene et al.,1991). Inasmuch as both B313 and B311 appear to survive in the skin forthe same time span, a possible explanation for these results ofimmunization was that Osp proteins are an important stimulus for thehost immune system to recognize the spirochete.

Polyclonal Antisera to B311 and B313

The lack of an antibody response to B311 and other Osp-bearing cells bymice immunized with B313 might also be explained by the presence ofunique antigens in B313 cells. According to this hypothesis, antibodieswere produced in response to live cell immunization with B313 but theywere directed against antigens found only in B313 cells. There have beenreports indicating that B. burgdorferi has other surface proteinaceousantigens that those been defined as Osps (Brandt et al., 1990; Katona etal., 1992; Luft et al., 1989; Sambri et al., 1991; Simpson et al.,1991). These considerations suggested the possibility of non-Ospantigens' being present on the surface of the mutant cells.

TABLE 3 INTRADERMAL IMMUNIZATION AND PROTECTION OF MICE WITH LIVE B311AND B313 Immunogen^(a) Mouse No ELISA^(b) GIA^(c) Experimentalinfection^(d) B311 1 256 1024 0/5 2 128 128 3 256 512 4 256 512 5 128128 B313 1 4 <16 5/5 2 8 <16 3 4 <16 4 4 <16 5 8 <16 Control^(e) 1 4 <165/5 2 2 <16 3 4 <16 4 4 <16 5 4 <16 ^(a)10⁶ cells were injectedintradermally in the abdominal region of each mouse at day 0.^(b)Reciprocal ELISA titers of individual mouse sera against Sh.2 cellsat day 24. ^(c)Reciprocal growth inhibition titers of individual mouseserum with 2 HU of guinea pig complement against Sh.2 cells at the day24. ^(d)Syringe challenge with 10⁴ B. burgdorferi strain Sh.2 wasperformed at the day 28 (number of mice infected/total tested).^(e)Control mice were injected with solution of 0.1X BSK II in PBS.

Previous studies have shown that there is little detectable antibodyresponse after live cell intradermal immunization with Osp-less cells ata dose that evokes antibodies in animals immunized with Osp-bearingcells. Consequently, to study the immunogenicity of Osp-less cellsanother immunization approach was needed. Mice were immunized with B311and B313 whole cell emulsified in an adjuvant and boosted once with thesame preparation. Sera were examined against both immunogens 7 wk afterthe initial immunization; the results are presented in Table 4. Firstexamined was the immune response by dry ELISA; it was found thatreciprocal titers for a homologous reaction were as high as 32,768. Whenheterologous sera were evaluated, the reciprocal titers were still high:16,384 for anti-B311 serum against B313 cells, and 4,096 for anti-B313serum against B311 cells. Sera from mice immunized with CFA alone werenegative at a dilution of 1:2. These results confirmed that, besidesknown Osps, there were other immunogenic components recognized by mice.

Antisera pooled from within the same group were also evaluated by GIAfor functional activity (Table 4). To avoid the deleterious effect ofcomplement on Osp-less cells the serum was heat-inactivated. Thereciprocal growth inhibitory titer of anti-B311 against B311 was high at8,192. Anti-B313 serum did not effect B311 cells at any of the dilutionsexamined. Moreover, Osp-less mutant cells were inhibited by anti-B311polyclonal serum only at a dilution of 1:32. The latter result, whileindicating the specificity of the response, nevertheless, suggested thatgrowth inhibitory antibodies to non-Osp components were produced. Thiswas confirmed by examining the Osp-less mutant cells with homologousanti-B313 serum the reciprocal growth inhibitory titer was 4,096. Therewas not growth inhibition of either B311 or B313 cells by sera of miceimmunized with adjuvant and PBS alone.

mAbs Against the Osp-less Mutant

To further characterize the surface antigens of the Osp-less mutant mAbsto B313 were produced. Procedures used for production and screening ofhybridoma supernatant fluids were designed to select for and identifythose mAbs that were directed against surface proteins and hadfunctional activity by GIA. To enhance selection of antibodies againstsurface components mice were boosted intravenously with live B313 beforethe spleen fusion. As a screen for surface-directed mAbs, an ELISA wasused in which whole borrelias were not dried in the microtiter platewells. To further evaluate mAbs for surface binding all hybridomasupernatants identified by wet ELISA were examined by unfixed cellimmunofluorescence assay. Using these assays several mAbs specific forB313 cells were identified.

Six mAbs produced against the Osp-less mutant were selected for furtherstudy by Western blot and GIA. Two different classes of mAbs weredistinguished and designated A and B, in the screening by unfixed cellIFA. The 3 class A mAbs produced prominent cell blebs and 4+ cellaggregates; the 3 class B mAbs produced 3+ aggregates and did notproduce blebs (FIG. 1). The morphologic changes observed with class AmAbs were similar to what was observed when bactericidal antiborelialantibodies were used (Coleman et al., 1992; Sadziene et al., 1994).Class A mAbs were associated with a homogeneous patchy pattern ofbinding to whole cells and little fluorescent staining of thebackground.

In contrast class B mAbs in the wet IFA did not produce staining ofsingle whole cells. Instead it was associated with numerous fluorescentspots in the background. By GIA class A antibodies were inhibitory atdilutions of hybridoma supernatant of 1:256-2048; class B mAbs inhibitedgrowth only at dilutions of supernatants of 1;16 or lower. Both class Aand B mAbs inhibited the growth of B311 at a dilutions of 1:16 or 1:32,but not at

TABLE 4 ANALYSIS OF POLYCLONAL MOUSE ANTISERA TO B311 AND B313 CELLS BYELISA AND GROWTH INHIBITION ASSAY^(a) Polyclonal ELISA^(b) Growthinhibition assay^(c) serum Mouse No B311 B313 B311 B313 Anti-B311 116384 16384 8192 32 2 16384 16384 3 32768 16384 4 32768 16384 Anti-B3131 4096 16384 <8 4096 2 4096 16384 3 4096 32768 4 2048 32768 Control^(d)1 <4 <4 <8 <8 2 <4 <4 ^(a)Mice were immunized with B311 and B313 wholecell emulsion in CFA and were boosted once with the same immunogen.^(b)Reciprocal ELISA titers from individual mouse sera. ^(c)Reciprocalgrowth inhibitory titers of heat-inactivated (56° C., 30 min) pooledmouse sera. ^(d)Control mice were immunized with complete Freund'sadjuvant emulsion in PBS.

higher dilutions. None of the antibodies inhibited the growth of B.hermsii. When 1 HU of guinea pig complement was added, it did notincrease the inhibitory effect of either class of mAb against B313cells.

Western Blot Analysis of mAbs

The two classes were also distinguishable by Western Blot. Class B mAbsdid not bind to any protein in the blots, a result that suggested theremAbs were directed against conformational epitopes or non-proteinaceousantigens. In contrast, all class A mAbs were reactive by Western blotand bound to the same low molecular weight protein. The results with twoclass A mAbs, 15G6 and 7D4, are shown in FIG. 2. Both these class A mAbswere IgG2b. An OspA⁻OspB⁻ B. burgdorferi mutant of HB19 lineage has beendescribed that expressed a surface protein not detectable in theosp-bearing wild-type population (Sadziene et al., 1992). Therefore, itwas determined whether or not other lineages of B31 express the proteinrecognized by 15G6 and 7D4 mAbs. An antibody-reactive protein with anM_(r) of 13,000 was present in all the B31 cell lineages investigatedand in similar amounts. This protein was designated “p13” and was boundby both mAbs. Identically-sized proteins bound by 15G6 and 7D4 werepresent in HB19 and Sh2 strains as well (data not shown). Both mAbs alsoproduced minor bands with proteins with M_(r)'s of 26,000, 32,000, and44,000 (FIG. 2).

Next, it was determined whether 15G6 or 7D4 mAbs recognized similar oridentical proteins in other genomic species of Lyme disease borrelias.The results with 15G6 are shown in FIG. 3; the same results wereobtained with 7D4. Representatives of B. afzelii and B. garinii wereevaluated at the same time as B311, B313 and B. hermsii cells by Westernblot. The mAb recognized a protein of slightly higher apparent molecularweight in B. afzelii ACAI. Neither 15G6 nor 7D4 recognized any proteinin B. garinii IP90 or B. hermsii.

It was also investigated whether p13 was cleaved from intact cells byproteinase K, as has been shown for other B. burgdorferi surfaceproteins (Bundoc and Barbour, 1989). No band was observed by Westernblot with either anti-p13 kDa mAb after proteinase K digestion ofwild-type and Osp-less mutant cells, an indication that p13 issurface-exposed. The result with mAb 15G6 and B313 cells is shown in theright panel of FIG. 3.

Immunofluorescence Studies of p13

To further assess the topography of p13 in the cell, in particular todetermine if p13 is exposed over B313's entire surface, fixed andunfixed cells were used in indirect (IFA) and direct (DFA)immunofluorescence assays. Purified 15G6 mAb was used; for unfixed cellDFA purified 15G6 mAb was conjugated with fluorescein.

In the fixed cell IFA B311 and B313 cells were individually mixed with asuspension of washed rat erythrocytes and coated as a thin smear overthe slides. No fluorescein-labeled spirochetes were seen with eitherwild-type or mutant cells when cells were exposed to 15G6 mAb. Incontrast, anti-flagellin mAb H9724, used as a control, showed uniformfluorescein labelling of fixed to the glass spirochetes, as described(Barbour et al., 1983). This suggested that the epitope for the 15G6 mAbwas sensitive to the experimental conditions and treatment required forthe sample preparation. Although this epitope was accessible to 15G6 mAbby the Western blot in the whole-cell lysates, it was not recognized inthe dried and fixed borrelias.

The binding of fluorescein-labeled antibodies to fixed and unfixedborrelias were assessed. B313 cells were examined at 3, 15, 30, 60, and360 min after addition of the 15G6 conjugate. The cells began tofluoresce within 3 min of addition of the conjugate; the antibody wasuniformly distributed over the length of the cell by 30 min (FIG. 4).Cells remained motile for up to 30 min. Cell aggregates and blebs becameevident after 15 min and increased in amounts over the 6 hours'observation (FIG. 4). In contrast to B313, very few (<1%) of B311 cellswere detectably bound by 15G6 conjugate by DFA with unfixed cells.

The finding that mAb 15G6 had some inhibitory activity against B313cells, albeit only at a low dilution, suggested that p13 of OspA⁺B⁺cells was accessible to some degree to the antibody. To determinewhether this putative exposure could be increased in wild-type cells bythe additional presence of an anti-Osp antibody in the suspension,anti-OspB mAb H6831 or anti-OspA mAb H5332 were used in combination withthe 15G6 conjugate. Both antibodies of the combination were added at thesame time. The 15G6 conjugate was also used by itself against B311 orB313. Immunofluorescence of cells was examined in 2 h.

As expected the conjugate by itself did not bind to B311 cells when usedalone. The conjugate produced aggregation and homogeneous staining ofcells of B313 (FIG. 5). In contrast, the binding of the 15G6 conjugateto B311 cells in the presence of the anti-OspA or anti-OspB mAbs was nothomogeneous. The results with H6831 and the conjugate are shown in FIG.5. The cells were found in large aggregates with patches of fluorescencedispersed throughout the clump of cells. The experiment showed thatsimultaneous exposure to mabs directed against OspA or OspB resulted inexposure of p13 protein by mAb 15G6.

Functional Characterization of Anti-p13 mAb

Results of these studies prompted further investigation of mAb 15G6 atthe functional level, using the whole purified IgG molecule andunivalent Fab fragments of 15G6 mAb. Two bactericidal antibodies,anti-OspB mAb H6831 and anti-Bh33 mAb. H4825 prepared in the same wayserved as controls (Sadziene et al., 1994). All mAbs were tested withB311 and B313 cells by GIA (Table 5). As reported previously, theanti-OspB mAb H6831 was highly effective in killing B311 cells but, asexpected, produced no damage on the Osp-less mutant cells. The effect of15 G6 mAb to the Osp-less cells, however, was marked. The MIC of thewhole IgG was 20 ng/ml. Univalent fab fragments inhibited growth at aconcentration of 200 ng/ml, the same as was observed with thebactericidal Fab fragment directed against Bh33, H4825 (Sadziene et al.,1993C).

As found previously with hybridoma supernatants, 15G6 in purified forminhibited growth of B311 cells only at 25 μg/ml or above. No growthinhibition with either B313 or B311 was observed with the anti-B.hermsii mAb H4825. This study proved the functional importance of thenewly identified 15G6 mAb to the Osp-less mutant cells and providedevidence that mabs active as Fab fragments can be produced against othersurface proteins besides Osp proteins.

Murine hybridomas secreting monoclonal antibody 15G6 were deposited withthe American Type Culture Collection, 10801 University Blvd., Manassas,Va. 20110-2209 under the terms of the Budapest Treaty on Mar. 18, 1999,under Deposit Numbers ATCC HB-12685 and ATCC HB-12686.

The combination of anti-Osp and anti-p13 mAbs on wild-type cells wasfurther characterized by GIA. Wild-type cells were is exposed totwo-fold serially diluted purified H6831 (anti-OspB), H5332 (anti-OspA),or, as a control, anti-Bh33 mAb H4825. 15G6 mAb was simultaneouslyapplied at 200 ng/ml, 10× the MIC for B313 and less than 0.01× the MICfor B311. GIA without the addition of mAb 15G6 was performed inparallel. The results are shown in Table 6. There was no effect with mAbH4825, with or without the addition of 15G6 mAb. There was only atwo-fold decrease in the MIC of mAb H5332 when mAb 15G6 was added. Theeffect of the addition of 16G6 to H6831 was more pronounced: without15G6, its growth inhibitory concentration was 150 ng/ml, whereas withaddition of 15G6, intensive cell blebbing occurred at concentrations64-128-fold lower, i.e., 1-2 ng/ml. These results were consistent withobservations of the combination by immunofluorescence assay and indicatethat anti-Osp and anti-p13 mAbs are synergistic in their activityagainst B. burgdorferi.

An isolate of B. burgdorferi lacking OspA, B, C, and D was characterizedwith respect to biological functions and its surface antigens, inparticular a 13 kDa protein. These results are likely also applicable toother strains of B. burgdorferi

TABLE 5 GROWTH INHIBITION BY PURIFIED WHOLE IgG AND Fab FRAGMENTS OFmAbs 15G6, H6831 and H4825 mAbs Minimal growth inhibitory concentration(μg/ml) 15G6 H6831 H4825 Whole Fab Whole Fab Whole Fab Cells IgGfragment IgG fragment IgG fragment B311 12.5 25 0.15 2 >25 >25 B313 0.020.2 >25 >25 >25 >25

TABLE 6 GROWTH INHIBITION BY PURIFIED WHOLE IgG OF mAbs H6831, H5332 ANDH4825 IN COMBINATION WITH mAb 15G6^(a) Minimal growth inhibitoryconcentration (ng/ml) H6831 H5332, H4825 Without 15G6 150 600 >25000With 15G6^(a) 1-2 300 >25000 ^(a)The amount of purified whole IgG of15G6 mAb was 10X MIC for B313 cells.

sensu lato and the other genomic species of Lyme disease agents. Otherisolates of Lyme disease borrelias have one or more of the Osp proteins(reviewed in Barbour and Fish, 1993). The study showed that the Osp-lessmutant differed in several ways from the OspAB-bearing parent with whichit was compared with. Although the most prominent structural differencebetween B311 and B313 was their Osp protein phenotypes, differences inother, less abundant proteins, or in non-proteinaceous components mayhave affected changes in function. The most apparent genetic differencebetween the OspA⁺B⁺ B311 and OspA⁻B⁻ B313 was the present or absence ofthe entire 49-kb linear plasmid.

Biological characteristics distinguishing Osp-less and Osp-bearing cellswas growth rate and the population density at which stationary phaseoccurred. Isolate B313 grew more slowly than did B311 and stoppeddividing at a lower cell density than did B311. This may be attributablewholly or in part to the greater auto-agglutination displayed by themutant cells. The triad of self-aggregation, slower growth rate, andlower cell density at stationary phase have also been noted withlow-passage, infectious isolates of B. burgdorferi (Barbour, 1984;Sadziene et al., 1993C). Like B313, some low-passage isolates of B.burgdorferi sensu lato also have a poor plating efficiency on solidmedium. The diminished ability of aggregated Osp-less borrelias to moveabout the broth medium may explain their slower growth under thatcondition, but why B313 cells could not grow on solid medium when singlydispersed is unknown. Low plating efficiency also is a feature of B314cells, which lack the 16-kb linear plasmid as well as the 49-kb plasmid(Sadziene et al., 1994). Inasmuch as B314 cells express OspC protein,the lower plating efficiency cannot be attributed to lack of Ospproteins per se.

Curiously, while OspA⁻B⁻ cells seem to be inherently more sticky for oneanother, they were less disposed than OspA⁺B⁺ cells to adhere to humanendothelial cells. This indicates that the phenomenon ofself-aggregation is not equivalent to the association of the borreliaswith mammalian cells. Prior studies had revealed functions for OspA inendothelial cell adherence and for OspB in cell penetration (Comstock etal., 1989; Comstock and Thomas, 1991; Thomas and Comstock, 1989). Thefindings of the present study are also consistent with a role for OspAand/or OspB in the association of borrelias with mammalian cells.

The present invention also examined another possible function of Ospproteins, namely resistance to non-immune effects of serum. For ablood-borne pathogen this would seem to be a requirement for successfultransmission between hosts and for proliferation within a mammalianhost. Much is known about what confers “serum-resistance” toGram-negative and Gram-positive bacteria; less is known about thisaspect of spirochetes. Although borrelias have two membranes sandwichinga peptidoglycan layer, as do Gram-negative bacteria, the outer membraneof borrelias appears to be more fluid than that of Gram-negativebacteria (Barbour and Hayes, 1986) and lack lipid A-containingglycolipids (Takayama et al., 1987). Thus, it was not likely a priorithat spirochetes would have a similar mechanism for avoiding thealternative complement pathway and other non-immune defenses againstbacteria. Indeed, the results suggest that OspA and/or OspB protect thecells from complement attack. When OspA, B, C, and D are lacking, theborrelias were more susceptible than OspA⁺B⁺ cells to unheated,nionimmune serum and to guinea pig complement.

Whatever protection OspA and OspB appeared to confer to the borrelias inserum did not seem to provide an advantage to cells in skin. In thesestudies two isolates were used that are not infectious by the criterionof detectable dissemination to the blood or other tissues. Surprisingly,the Osp-bearing cells did not survive for a longer period in the skinthan did their Osp-less counterparts. By 18 hours after inoculation bothB311 and B313 could not be recovered from skin samples placed in culturemedium. Infectious isolates persist in the skin for days (Barthold etal., 1991). The limited duration of survival noted in the present studymay also be a function of inherent strain differences. A non-infectiousisolate of strain HB19 of B. burgdorferi survived in the skin for 24hours by the same culture criterion.

Given the indistinguishability of B311 and B313 with respect to skinsurvival, one might expect that the immune responses to intradermalinoculation of viable borrelias would be comparable. Although theOsp-less mutant lacked two proteins, OspA and OspB, that areimmunodominant when syringe inocula of 10⁵ or greater are used (Bartholdet al., 1993; Gern et al., 1993; Greene et al., 1988; Roehrig et al.,1992; Schaible et al., 1993), other antigens, such as flagellin,commonly recognized by antibodies in immune sera were still present.Instead, it was found that there was little detectable immune responseto B. burgdorferi by ELISA, GIA, and infectious challenge when B313 wasthe immunogen.

Under the same conditions and with the same dose, mice given B311 hadhigh titers to B. burgdorferi by immunoassays and were protected againstchallenge with strain Sh.2. These results suggested that OspA and/orOspB not only are immunodominant antigens but also, perhaps throughtheir mitogenic properties (Ma and Weis, 1993), immunostimulatory.

The present invention also contemplates the possibility that there wereno antigens on the cell surface in B313 cells. Without Osp proteins, thecell surface of B. burgdorferi conceivably could be like Treponemapallidum's outer membrane, which is notably inert to the immune system(Radolf et al., 1989). To further assess this, mice were immunized withB313 and an adjuvant to enhance immune responsiveness. When this wasdone, the antiserum produced to B313 cells inhibited the growth ofhomologous cells but only minimally that of B311. The similar ELISAtiters for both anti-B311 and anti-B313 sera against homologous andheterologous cells indicated that with the appropriate adjuvant B313could elicit antibodies to antigens shared with B311. The GIA resultsshowed that there were unique features of the surface of B313 cells.These components were either not expressed by 311 cells or wereotherwise cloaked in these cells.

The minimal effectiveness of polyclonal anti-B311 sera in inhibiting thegrowth of B313 cells indicated that antibodies to OspA and/or OspBconferred growth inhibition.

The remaining antigens of the Osp-less mutant were further investigatedwith mAbs. The screening procedures were designed to identify antibodiesthat had the functional activity of growth inhibition. The antibodiesselected by this means fell into two classes: one in which theantibodies in broth medium produced large aggregates and prominentmembrane blebs and a second in which the antibodies produced smalleraggregates and minimal evidence of lysis. The first antibodies werefound to bind to a 13 kDa (p13) protein in Western blots. The secondgroup of antibodies did not bind to any component in blots. p13 and mAbsto it were therefore characterized in detail.

The evidence that the 13 kDa protein was surface-exposed in the Osp-lessmutant was the following: (i) agglutination of viable cells by antibody;(ii) growth inhibition by whole immunoglobulin and Fab fragment; (iii)direct immunofluorescent staining of live cells by an antibodyconjugate; and (iv) cleavage of antibody's epitope from the cell'ssurface by in situ treatment with protease. p13 was present in allmembers of the B31 lineage and in approximately equal amounts. Theexpression of the protein did not vary according the amount of one oranother the Osp proteins. A slightly larger protein recognized by themAb was present in a B. afzelii strain. If Ip90, a representative of B.garinii, have a homologous protein it does not share the mAbs' epitope.

It was considered whether p13 was identical to one of the other lowmolecular weight B. burgdorferi proteins to which antibodies have beendeveloped. Like antibody to p13, antibody to a 10 kDa protein, asreported (Katona et al., 1992), bound to only a small proportion ofOsp-bearing cells in immunofluorescence assays. However, the molecularsize of 10 kDa protein did not vary between strains and uniformfluorescein labeling was seen in fixed cell preparation when probed withmAb to 10 kDa protein (Katona et al., 1992). Furthermore, 15G6 does notbind to the 10 kDa in Western blots (Habicht, 1993). The presence of a14 kDa protein of B. burgdorferi was reported (Sambri et al., 1991).This was identified with a mAb and by immunofluorescence of liveborrelias. In contrast with what was observed with mAbs to p13 and withantibody to the 10 kDa protein (Habicht, 1993), antibody to the 14 kDaprotein of Sambri et al. bound to the majority of cells (Sadziene etal., 1993A). These differences suggest that p13 is neither the 10-kDanor 14-kDa proteins of B. burgdorferi.

The effect of 15G6 on susceptible borrelias was similar to what wasobserved with the anti-OspB mAb H6831 (Sadziene et al., 1994). Bindingto the cells was detectable by direct immunofluorescence by 3 minutes.The staining was homogenous and was followed by the appearance ofmembrane blebs and further cell aggregation even with Fab fragments. Theconcentration of 15G6 mAb at which growth inhibition and cell disruptionoccurred was 20 ng/ml. This was 10-fold lower to what was observed withH6381 mAb against B. burgdorferi and the same as with H4825 against B.hermsii (Sadziene et al., 1993C).

The failure of mabs to p13 to inhibit the growth of Osp-bearing cells isconsistent with lack of surface exposure of the protein, or at leastimpairment of the antibody's access to its target. The cloaking orobstruction could be from OspA, OspB, or a complex of the two. It wasalso possible that p13 was not in the outer membrane at all in B311cells; in those cells it may have been in the periplasmic space or inthe cytoplasmic membrane. Evidence against this latter possibility was(i) cleavage of anti-p13 mAbs epitopes from Osp-bearing cells by in situtreatment with proteinase K, (ii) the finding that when mAb 15G6 to p13was added to antibodies to either OspA or OspB, the growth inhibitoryconcentration for the anti-Osp antibodies was decreased, substantiallywith the bactericidal H6831 to OspB.

By itself mAb 15G6 had no discernible effect against B311 cells exceptat high concentrations. The finding suggested that p13 was exposed tomAb 15G6 when antibodies to OspA or OspB gathered together Osp proteinsin patches in the fluid outer membrane (Barbour et al., 1983). Theimmunofluorescence provided visual evidence of this; large membraneblebs of B311 cells treated with anti-OspA or -OspB proteins were boundby conjugated 15G6 mAb. This in vitro synergism between the twoantibodies, one directed against an Osp protein and the other againstp13, suggests that p13 in combination with OspA or OspB may be usefulfor immunoprophylaxis against Lyme disease.

These results also provide evidence of the interaction of antibodies andborrelias and, in particular, those lacking the known Osp proteins. Thetarget or targets for the second class of mAbs remains to be determined.It is also possible that they also bind to p13 but that their epitopesare sufficiently conformation-dependent that Western blots would benegative. Alternatively, there may be other proteins or othernon-proteinaceous components in the outer membrane against which thesefunctional antibodies act.

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The above references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, areall specifically incorporated herein by reference.

What is claimed is:
 1. A pharmaceutically acceptable composition,comprising an isolated protien having a molecular weight of about 13kDa, as determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS/PAGE), that binds to the monoclonal antibody 15G6.2. The composition of claim 1, wherein the composition further consistsessentially of B. burgdoferi outer membrane proteins OspA, OspB, OspC orOspD.
 3. The composition of claim 1, wherein the isolated protein isobtained from Borrelia burgdorferi cells.
 4. The composition of claim 1,wherein the isolated protein is a recomibinant protein.